The primary goal of this application is to obtain a better understanding of transcriptional regulation of the mu opioid receptor gene (Oprm) through molecular biological means and transgenic techniques. Soon after the MOR-1 cDNAs were cloned, the promoter (El promoter) and structure (exons 1-4) of the original MOR-1 gene were identified and characterized. This was soon followed by the isolation of two splice forms, MOR-1A and MOR-IB. The diversity of the MOR-l gene was further illustrated by our recent identification of an additional nine exons, twelve splice variants and a novel promoter associated with one of the new exons (E11promoter). This application focuses on elucidating the detailed structure and function of the E11 promoter and its relationships with E1 promoter. The E11 promoter is located at about10 kb upstream of the original E1 promoter. Alternative splicing of the transcripts driven by the E11 promoter yield eight variants encoding a number of novel proteins as well as the original MOR-1 protein. Three of the variants regulated by the E11 promoter generate the original MOR-1 protein. Thus, this single protein can be generated by four splice variants of the mu opioid receptor gene under the control of two distinct promoters. Differential expression of the variant mRNAs in various brain regions revealed region-specific RNA processing. Neuronal promoter activities with a core promoter and a negative element have been suggested. The specific aims to achieve the overall goal are: (1) Identification of cis-acting elements in E11 promoter region through mutant analysis; (2) Isolation of DNA-binding proteins that regulate promoter activities; (3) Investigation of the E11 promoter and its relationships with E1 promoter in transgenic mice. My long-term goal is to understand the mechanisms by which the MOR-1 gene is regulated and to gain insights into the pharmacological and physiological significance of its regulation. The significance of the E11 promoter and its associated variants is suggested by antisense mapping studies in which two antisense probes targeting exon 11 blocked spinal morphine and supraspinal M6G analgesia. The knowledge obtained from the application will help to determine the complexity and functional importance of MOR-l gene regulation, establish the cell models and the transgenic animal models for studying its gene regulation, and provide potential targets for developing novel drugs useful in pain control and drug abuse.